L-cystine dihydrochloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 September 2016 - 05 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

L-cystine dihydrochloride

EC Number:

250-391-9

EC Name:

L-cystine dihydrochloride

Cas Number:

30925-07-6

Molecular formula:

C6H12N2O4S2.2ClH

IUPAC Name:

L-cystine dihydrochloride

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

SkinEthic™ RHE-model RHE/S/17
- Batch no.: 16-RHE-114
- Expiration date: 07 November 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ambient temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with minimum 25 mL DPBS; excess DPBS removed by shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Observable damage in the tissue due to washing: no data
- Modifications to validated SOP: none

DYE BINDING METHOD
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin category 2 if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg ± 2 mg per tissue

NEGATIVE CONTROL
- Volume applied: 16 µL ± 2 µL per tissue

POSITIVE CONTROL
- Volume applied: 16 µL ± 2 µL per tissue (5 % solution in deionized water)

Duration of treatment / exposure:

42 minutes (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

The test item as well as the positive and negative control were tested in batch-triplicates.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

replicate 1

Run / experiment:

1

Value:

28.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

replicate 2

Run / experiment:

1

Value:

29.27

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

replicate 3

Run / experiment:

1

Value:

30.77

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1 Optical density and Tissue viability

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	viability	OD	viability	OD	viability	OD	viability	viability
Negative Control	1.925	98.52%	1.978	101.22%	1.959	100.26%	1.954	100.00%	1.37%
Positive Control	0.029	1.48%	0.028	1.45%	0.030	1.52%	0.029	1.48%	2.30%
Test item	0.557	28.50%	0.572	29.27%	0.601	30.77%	0.577	29.51%	3.91%

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of the present study, the test item is considered to possess an irritant potential to skin (UN GHS: Category 2).

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model.

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

After treatment with the negative control (DPBS-buffer) the mean OD was 1.954 (study acceptance criterion: > 1.431). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.48% (study acceptance criterion: < 3.14%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 29.51% and, thus, lower than 50%, i.e.according to OECD 439 the test item is considered as irritant to skin (UN GHS: Category 2).